Photobleaching techniques are used as a strategy to reveal the
diffusion/binding dynamics of molecules or the exchange between
compartments in live cells, tissues or organisms. The most
straightforward photobleaching technique is the Fluorescence Recovery
After Photobleaching (FRAP). In FRAP, fluorescent molecules are
deactivated in a region of interest by focused light (tipically a laser
beam) and the recovery of the fluorescence intensity in that region is
followed in time, quantified by suitable numerical analysis (normalization and curve
fitting), and compared to the initial conditions.
Fluorescence recovery into the region is due to the diffusion of the molecules in and out the region. Bleached molecules, which can diffuse out of the region, are substituted by other unbleached molecules that diffuse inside the region from surrounding areas. Quantification and analysis of fluorescence recovery provide information of descriptive parameters such as mobile and immobile fraction and half time of recovery.
Further analysis by kinetic modelling provides information on the molecular dynamics such as the diffusional properties of the studied molecule and its binding characteristics.